1. Field of the Invention PA1 (1) An antibody which specifically binds to human BTC protein or a mutein thereof; PA1 (2) The antibody according to (1) wherein human BTC protein has an amino acid sequence of SEQ ID NO:1; PA1 (3) The antibody according to (1) or (2) which is a monoclonal antibody; PA1 (4) The antibody according to any one of (1) to (3) which has no cross-reactivity with at least one of human epidermal growth factor, human transforming growth factor a and mouse BTC protein; PA1 (5) A monoclonal antibody which specifically binds to human BTC protein and is capable of neutralizing the biologial activity of said protein, which does not have cross-reactivity with human epidermal growth factor, human transforming growth factor a and mouse BTC protein, and which belongs to the immuno-globulin class of IgG; PA1 (6) The antibody according to anyone of (1) to (5) which recognizes an amino acid sequence of 31st (Arg) to 80th (Tyr) of SEQ ID NO:1; PA1 (7) A cloned hybridoma which is composed of a mammalian spleen cell immunized by human BTC protein or a mutein thereof and a homogenic or heterogenic lymphocyte; PA1 (8) A method of producing the monoclonal antibody of (5), which comprises proliferating the hybridoma according to (7) in a liquid culture medium or in an abdominal cavity of a mammal to form and accumulate the monoclonal antibody and then collecting the monoclonal antibody; PA1 (9) A method of detecting and assaying a human BTC protein or a mutein thereof which comprises contacting the antibody according to anyone of (1) to (6) with a specimen; PA1 (10) A method of diagnosing diabetes or complications thereof which comprises contacting the antibody according to anyone of (1) to (6) with a specimen and assaying a human BTC protein or a mutein thereof in the speciman; PA1 (11) A pharmaceutical composition which comprises an effective amount of the antibody according to anyone of (1) to (6) and a pharmaceutically acceptable carrier, excipient or diluent; PA1 (12) The pharmaceutical composition according to (11) for diagnosing diabetes or complications thereof; PA1 (13) A kit for diagnosing diabetes or complications thereof which comprises an effective amount of the antibody according to anyone of (1) to (6); PA1 (14) A kit for detecting and assaying a human BTC protein or a mutein thereof which comprises an effective amount of the antibody according to anyone of (1) to (6); and PA1 (15) A method of purifying a human BTC protein or a mutein thereof which comprises contacting the antibody according to anyone of (1) to (6) with a crude sample containing the protein or a mutein thereof and isolating it.
The present invention relates to an antibody to a human betacellulin (hereinafter also referred to as "BTC") protein or a mutein thereof, a hybridoma, their production and use thereof.
2. Description of the Preferred Embodiments
As to the family of epidermal growth factors, since epidermal growth factor (EGF) was discovered by S. Cohen, J. Biol. Chem., 237, 1555 (1962), Dev. Biol., 12, 394 (1965), various factors have been discovered. These factors are considered to have various functions such as differentiation, maturation, survival, functional retention and proliferation of not only epidermal cells, but also of various cells. Specifically, these factors include transforming growth factor .alpha. (TGF-.alpha.) [G. J. Todaro and J. E. DeLarco, Nature, 264, 26 (1976) and H. Marquardt et al., Proc. Natl. Acad. Sci. USA, 80, 4684 (1983)], amphiregulin [M. Shoyab et al., Science, 243, 1074 (1989)] and heparin-binding EGF (HB-EGF) [S. Higashiyama et al., Science, 251, 936 (1991)], in addition to EGF described above.
Human BTC protein is described in EP-A 0555785, and reported in Biochem. Biophys. Res. Commun., 190, 1173-1179 (1993). BTC protein was discovered as a novel cell growth factor produced by a transgenic mouse-derived pancreatic beta tumor cell [Y. Shing et al., Science, 259, 1604 (1993)]. Further, based on the nucleotide sequence of the gene thereof, the human BTC gene was cloned, and the characteristics of BTC protein were studied. As a result, from these studies, the following characteristics were discovered. (1) A precursor molecule having a transmembrane domain is processed at the C-terminus to produce matured type BTC protein, protein. (2) The BTC gene is expressed in the normal mouse kidney, lung, liver and pancreatic beta cells. (3) BTC protein exhibits growth promoting activity on a vascular smooth muscle cell and a retinal pigment epithelial cell. (4) BTC protein acts on an EGF receptor as a ligand [T. Watanabe et al., J. Biol. Chem., 269, 9966 (1994)]. From these, it is conceivable that BTC protein plays an important role in various organs, and abnormal expression thereof possibly induces diseases. Further, these results suggest the possibility that BTC protein contributes to the growth of smooth muscle cells relating to the crisis and progress of arterial sclerosis, and particularly to the possibility that BTC protein is involved in the crisis of diabetic vascular complications such as diabetic arterial sclerosis and diabetic retinitis or in the canceration of cells.
An antibody inhibiting BTC protein activity is considered to be used as one therapeutic agent for diseases in which BTC protein is involved. However, such a report which relates to an antibody to a human BTC Protien has not been presented till now. The assay of the human BTC protein level in the body fluids is also considered as one means for diagnosing the diseases as described above. However, it has not yet been achieved.
As described above, human BTC protein may participate in the crisis and progress of arterial sclerosis, and particularly in the crisis of diabetic vascular complications or the canceration of cells by its abnormal expression (excess expression). Accordingly, inhibition of excess activity of human BTC protein can conceivably treat these diseases. Therefore, the measurement of human BTC protein level in blood makes it possible to diagnose these diseases. In order to assay human BTC protein existing only in slight amounts in the body fluids, an assay with high sensitivity is required.